**2. Sampling methods**

50 New Approaches to the Study of Marine Mammals

immunosuppression and iii) genotoxicity.

(DDT) and its metabolites [17, 18].

contaminants, it is known that some contaminants, such as organochlorines (OCs), polybrominated diphenyl ethers (PBDEs), bisphenol A (BPA) and phthalates are endocrine disrupting chemicals (EDCs) and immunosuppressors [6, 7, 8, 9, 10]. Others, such as polycyclic aromatic hydrocarbons (PAHs), derived from both natural (e.g., oil spills, forest fires, natural petroleum seeps) and anthropogenic (e.g., combustion of fossil fuels, use of oil for cooking and heating, coal burning) sources, are carcinogenic, teratogenic and mutagenic compounds [11] and some studies have shown that PAHs with four or more rings can induce dioxin-like activity and weak estrogenic responses [12]. Moreover PAHs have attracted scientific interest due to their genotoxicity [13]. But how is it possible to discriminate the effects of a specific toxic in a mixture of many pollutants and assess the susceptibility of a particular cetacean species to just one class of contaminants? The aim of the present study is to use cetacean fibroblast cell cultures, obtained from skin biopsy of free-ranging animals and from skin tissue of stranded animals dead within 12 h [14, 15], as an "in vitro" method, called "*Test Tube Cetaceans*", to investigate the effects of environmental contaminants. In particular we use *Test Tube Cetaceans* to explore the susceptibility to genotoxic effects of different environmental contaminants in these marine mammals. Cell cultures were obtained from several species of cetaceans: fin whale (*Balaenoptera physalus*) and Bryde's whale (*Balaenoptera edeni*) for mysticetes, sperm whale (*Physeter macrocephalus*), killer whale (*Orcinus orca*), Risso's dolphin (*Grampus griseus*), bottlenose dolphin (*Tursiops truncatus*), striped dolphin (*Stenella coeruleoalba*), long-beaked common dolphin (*Delphinus capensis*) and common dolphin (*Delphinus delphis*) for odontocetes. Here we present the results for three different biomarkers of anthropogenic stress in cetacean cell cultures that will enable us to assess: i) exposure to contaminants, ii)

i. **Interspecies differences in the mixed function oxidase (MFO) induction as biomarker of exposure to different environmental contaminants:** the evaluation in fibroblast cell cultures with immunofluorescence technique of the presence and the induction of two components (CYP1A1 and CYP2B) of the cytochrome P450 monooxygenase system (MFO), among the most relevant in drug and xenobiotic metabolism, was used to evaluate interspecies sensitivities to various classes of environmental contaminants. In particular CYP1A1 is induced by planar compounds such as planar OCs (coplanar polychlorinated biphenyls (PCBs)) and PAHs [16] and CYP2B by globular compounds such as PBDEs, PCBs and OC insecticides such as dichlorodiphenyltrichloroethane

**ii. Qualitative and quantitative major histocompatibility complex (MHC) class I chain related protein A (MICA) expression as toxicological stress marker of the immune system:** the evaluation of the qualitative and quantitative MICA protein expression in fibroblast cell cultures with the immunofluorescence technique was used as toxicological stress marker of the immune system of different species of cetaceans [19]. The genes encoding for MICA and MICB are found within the major histocompatibility complex. Although MIC products have been found in various cells/tissues, the current consensus is that MIC genes are mainly expressed in gastrointestinal epithelium,
